title: Next generation sequencing of viral RNA genomes.
abstract: BACKGROUND
  With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform.
  
  
  RESULTS
  As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed.
  
  
  CONCLUSIONS
  The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.
authors.forename: Denise A
authors.initials: DA
authors.lastname: Marston
authors.forename: Lorraine M
authors.initials: LM
authors.lastname: McElhinney
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ellis
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Horton
authors.forename: Emma L
authors.initials: EL
authors.lastname: Wise
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Leech
authors.forename: Dan
authors.initials: D
authors.lastname: David
authors.forename: Xavier
authors.initials: X
authors.lastname: de Lamballerie
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Fooks
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000821:virology; D002460:Cell Line; D006224:Cricetinae; D018740:Genetic Heterogeneity; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D018114:Lyssavirus / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D012367:RNA, Viral / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D014776:Virus Cultivation
chemical_list: D012367:RNA, Viral